Abstract: Somatic Cell Evolution in Small Human Replicative Units This Project studies fundamental parameters of evolution (mutation, drift, and selection MDS) in normal human and animal cells. Although MDS underlies somatic cell evolution, very little is understood about these parameters because they are difficult to study in humans. We will translate methods from evolutionary biology into systems biology. We will study MDS in distinct, small replicative units (intestinal crypts). The compartmentalization of cells into small replicative units can modify evolution because selection and drift (random cell turnover) is limited to immediately adjacent cells. The advantages of analyzing small replicative units are that experimentally they large enough to measure with conventional methods yet small enough to simulate in detail. Characterizing somatic cell evolution in replicative units can lead to better understanding of tumor evolution because selection or drift occurs between neighboring cells. We will better characterize MDS in crypts based on our existing published (Shibata, Graham) human crypt simulations. These simulations have already inferred stem cell numbers and dynamics based on smaller amounts of data. The new sequencing data are a richer resource because more MDS parameters are encoded by mutations (mutation rates and mechanisms, dN/dS, passenger versus driver, neoantigen accumulation). The sequencing data is augmented by crypt epigenetic and expression data to more fully characterize normal somatic cell evolution. We will sample 8 colon and small intestinal crypts from 40 different aged individuals. For each crypt, we will measure mutations (whole genome sequencing), epigenetic alterations (ATAC-seq), and expression (NanoString). We will also measure APC+/- crypts to determine whether somatic cell evolution changes after a gatekeeper mutation. We will also measure crypt somatic cell evolution in mouse and elephant crypts to determine if their evolution differs. We will also determine the DNA damage and stress response in fibroblasts from 57 different mammalian species. We will determine de novo mutation rates across the same 57 mammalian cell lines through expansion of single fibroblast cells followed by deep sequencing. We will correlate mutation rates with cancer rates in the same mammalian species as determined in Project 1. Finally, using prioritized candidate gene lists generated in Project 1, we will perform gene editing experiments on the top 5 genes from different species most likely to contribute to their evolution of cancer resistance. The significance of these studies is a better characterization of basic MDS evolution parameters. The species studies will provide perspective on whether MDS parameters are fixed or can vary with age or species, identifying which parameters are more amenable for intervention. A complete systems biology solution of normal human crypts will facilitate further efforts with much larger groups of somatic cells. This Project will lay the groundwork for future work to impact patient care through evolutionary-based solutions to cancer.